Constitutively active histamine H3 receptor mutants and uses thereof

ABSTRACT

Internal domain 3 of seven transmembrane G protein-coupled receptors is important for G protein binding or receptor activity, and is well conserved. In H3 receptors, which are a type of G protein-coupled receptor, this region is also conserved in the same way. Therefore, as a result of using PCR to introduce point mutations into sequences encoding the region in H3 receptor cDNA, H3 receptor mutants comprising extremely strong constitutive activity could be successfully produced. The present inventors further found that by using constitutively active H3 receptor mutants, drug candidate compounds such as H3 receptor inverse agonists can be screened more easily and efficiently.

TECHNICAL FIELD

The present invention relates to constitutively active histamine H3receptor mutants and uses thereof.

BACKGROUND ART

Many hormones and neurotransmitters regulate body functions throughspecific receptors present on the cell membrane. Many of these receptorstransmit signals into cells by activating conjugating guanosinetriphosphate-binding proteins (G proteins). These receptors aretherefore generically referred to as G protein-coupled receptors(GPCRs). Alternatively, since they also share a structure comprising aseven membrane-permeating region, they are also generically referred toas ‘seven transmembrane’ receptors.

G protein-coupled receptors are present on various functional cellsurfaces in cells and organs of the body, and play extremely importantroles as targets of molecules such as, for example, hormones,neurotransmitters, and physiologically active substances that regulatethe functions of these cells and organs of the body. Consequently, Gprotein-coupled receptors have been attracting considerable attention astargets of drug development. A number of G protein-coupled receptors areknown to be constitutively active (Costa, T. et al., Mol Pharmacol, 41,549-560, 1992; Lefkowitz, R. et al., Trends Pharmaco. Sci., 14, 303-307,1993). In some cases when a mutation is introduced into Gprotein-coupled receptors, their activity is known to further increase.For example, a constitutively active mutant of the a1B-adrenalinereceptor, a type of G protein-coupled receptor, is known (Kjelsberg, M.A. et al., J. Biol. Chem. 267, 1430-33, 1992). Additionally, WO 01/77172discloses constitutively active mutants of various G protein-coupledreceptors.

In addition, antagonists that exhibit actions opposite to agonist haverecently been discovered, indicating that the inverse agonists may bedrug candidate compounds targeting G protein-coupled receptors(Milligan, G. et al., Trends Pharmaco. Sci., 16, 10-13, 1995). Wheninverse agonists act on G protein-coupled receptors, a conformationchange arises, which is thought to increase the proportion of inactiveforms (Milligan, G. et al., Trends Pharmaco. Sci., 16, 10-13, 1995).

Histamine H3 receptors (H3 receptors) are known to be a type of Gprotein-coupled receptor. Genes that encode these receptors are reportedto exist in various living organisms, such as humans (Lovenberg, T. W.et al., Molecular Pharmacology, 55: 1101-1107, 1999; Lovenberg, T. W. etal., Journal of Pharmacology and Experimental Therapeutics, 293:771-778, 2000; Tardivel-Lacombe, J. et al., Molecular Neuroscience, 11:755-759, 2000; WO 2003004637). H3 receptor gene knockout mice have beenfound to demonstrate increased body weight, food intake and bloodinsulin or blood leptin levels, thus clearly indicating a correlationbetween H3 receptors and diseases characterized by changes in bodyweight, food intake and blood insulin or blood leptin levels (WO2003004637). Furthermore, H3 receptors are constitutively active, evenin their natural states, and have been reported to easily adoptconstitutively active conformations (Morisset, S. et al., Nature, 408,860-864, 2000). However, to date there have been no reports of examplesof constitutively active H3 receptor mutants.

DISCLOSURE OF THE INVENTION

In consideration of the aforementioned circumstances, the object of thepresent invention is to produce constitutively active H3 receptormutants, and to provide methods of screening for drug candidatecompounds using these constitutively active mutants.

The present inventors conducted extensive research to solve theaforementioned problems. Internal domain 3 of the seven transmembrane Gprotein-coupled receptors is important for G protein binding or receptoractivity, and is well conserved. In H3 receptors, which are a type of Gprotein-coupled receptor, this region is also conserved in the same way.Therefore, an attempt was made to produce constitutively active H3receptor mutants. First, PCR was used to introduce point mutations intosequences that encode this conserved region in mouse H3 receptor cDNA,thus producing clones MT1, MT2, MT3, MT5, and MT6. Next, wild type mouseH3 receptor cDNA and five mouse H3 receptor mutant cDNAs wererespectively transfected into cell line HEK293. cAMP levels were thenmeasured using ELISA. As a result, cAMP levels in all clones were foundto decrease in a histamine dose-dependent manner in the presence of 10μM forskolin. In addition, cAMP levels were found to increase withincreased doses of thioperamide, an H3 inverse agonist, in the presenceof 10 μM forskolin. Moreover, with the exception of the MT1 clone, cAMPlevels were found to be increased compared to natural H3 receptors. Theabove results indicate that the present inventors had succeeded inproducing H3 receptor mutants comprising extremely strong constitutiveactivity. In addition, by using constitutively active H3 receptormutants, screening of drug candidate compounds such as H3 receptorinverse agonists was shown to be possible with more ease and efficiency.

More specifically, the present invention provides the following:

-   [1] a constitutively active H3 receptor mutant;-   [2] the constitutively active mutant of [1], wherein at least one    amino acid residue of the activation-regulating site on the    C-terminal side of H3 receptor internal domain 3 is substituted with    another amino acid residue;-   [3] the constitutively active mutant of [1] or [2], wherein an amino    acid residue of a site corresponding to at least one of amino acid    352, 353, 354, or 357 in the amino acid sequence of SEQ ID NO: 1 or    SEQ ID NO: 3 is substituted with another amino acid residue;-   [4] the constitutively active mutant of [1] or [2], wherein the    substitution of an amino acid residue in the H3 receptor activation    regulating site is either (a) or (b) below:    -   (a) a substitution from RDRKVAK (SEQ ID NO: 11) to KDHKVLK (SEQ        ID NO: 4), RARKVAK (SEQ ID NO: 5), RDRKVIK (SEQ ID NO: 6) or        RDRKVKK (SEQ ID NO: 7) in a human H3 receptor; or    -   (b) a substitution from RDKKVAK (SEQ ID NO: 12) to KDHKVLK (SEQ        ID NO: 4), RAKKVAK (SEQ ID NO: 8), RDKKVIK (SEQ ID NO: 9) or        RDKKVKK (SEQ ID NO: 10) in a mouse, rat, or guinea pig H3        receptor;-   [5] the constitutively active mutant of [1] or [2], comprising an    amino acid substitution of at least one of (a) to (c) below:    -   (a) at least a substitution from A to K or I at amino acid 357        in the amino acid sequence of SEQ ID NO: 1;    -   (b) at least a substitution from D to A at amino acid 353 in the        amino acid sequence of SEQ ID NO: 1; and    -   (c) at least a substitution from R to K at amino acid 352, K to        H at amino acid 354, and A to L at 357 in the amino acid        sequence of SEQ ID NO: 1;-   [6] the constitutively active mutant of [1] or [2], comprising an    amino acid substitution of at least one of (a) to (c) below:    -   (a) at least a substitution from A to K or I at amino acid 357        in the amino acid sequence of SEQ ID NO: 3;    -   (b) at least a substitution from D to A at amino acid 353 in the        amino acid sequence of SEQ ID NO: 3; and    -   (c) at least a substitution from R to K at amino acid 352, K to        H at amino acid 354, and A to L at amino acid 357 in the amino        acid sequence of SEQ ID NO: 3;-   [7] a DNA encoding the constitutively active mutant of any one of    [1] to [6];-   [8] a vector inserted with the DNA of [7];-   [9] a transformed cell comprising the DNA of [7] or the vector of    [8];-   [10] a method for evaluating whether or not a test compound changes    the activity of a constitutively active H3 receptor mutant, wherein    the method comprises:    -   (a) contacting the test compound with cells expressing the        constitutively active H3 receptor mutant; and    -   (b) detecting the activity of the constitutively active mutant        in the cells,    -   wherein the test compound is judged to change the activity of        the constitutively active mutant when the activity increases or        decreases compared with that in the absence of the test        compound;-   [11] the method of [10], wherein the activity of the constitutively    active mutant is detected by using a change in cAMP concentration, a    change in calcium concentration, a change in G protein activity, a    change in phospholipase C activity, or a change in pH, as an    indicator;-   [12] a method of screening for a drug candidate that changes the    activity of a constitutively active H3 receptor mutant, wherein the    method comprises steps (a) and (b) below:    -   (a) using the method of [10] or [11] to evaluate a number of        test compounds to determine whether or not they change the        activity of a constitutively active H3 receptor mutant; and    -   (b) selecting from the number of test compounds a compound(s)        judged to change the activity of the constitutively active        mutant;-   [13] the method of [12], wherein the drug candidate is an H3    receptor inverse agonist.

H3 receptors are known to have constitutively active forms. However, thepresent inventors produced constitutively active H3 receptor mutantscomprising even higher activity than natural constitutively activeforms. The present inventors also found that drug candidate compoundscan be screened more easily and efficiently by using theseconstitutively active mutants. The present invention is based on thesefindings.

The present invention provides constitutively active H3 receptormutants. Constitutively active H3 receptor mutants in the presentinvention are preferably substantially purified. In the presentinvention, “substantially purified” refers to being isolated from theexternal environment such that other components account for at most 40%,preferably 25%, and more preferably 10% or less. In addition, in thepresent invention, “constitutive activity” refers to activity in theabsence of ligands (i.e., states in which activity exists even whenligands are absent).

The types and number of mutated sites in the constitutively activemutants of the present invention are not particularly limited, however,the mutated sites are preferably located at the activation regulationsite on the C-terminal side of H3 receptor internal domain 3. Examplesof mutation types include substitution mutations, deletion mutations,and insertion mutations; however, substitution mutations are preferable.Examples of constitutively active mutants comprising this type ofmutation are those constitutively active mutants in which at least oneamino acid residue at the activation regulation site on the C-terminalside of H3 receptor internal domain 3 is substituted with another aminoacid residue. More specifically, examples of constitutively activemutants are those comprising an activation regulation site that includesthe sequence KDHKVLK (SEQ ID NO: 4), RARKVAK (SEQ ID NO: 5), RDRKVIK(SEQ ID NO: 6), RDRKVKK (SEQ ID NO: 7), RAKKVAK (SEQ ID NO: 8), RDKKVIK(SEQ ID NO: 9), or RDKKVKK (SEQ ID NO: 10), however, the sequence of theactivation regulating site in a constitutively active mutant of thepresent invention is not limited to these sequences.

In addition, other examples of constitutively active H3 receptor mutantsof the present invention include, but are not limited to, constitutivelyactive mutants in which an amino acid residue at a site corresponding topositions 352, 353, 354, or 357 in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 3, is substituted with another amino acid residue. Forexample, constitutively active mutants in which mutations have occurredat another site are also included in the constitutively active mutantsof the present invention, in addition to those described above.

In the present invention, examples of a site that corresponds topositions 352, 353, 354, or 357 in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 3 include positions 352, 353, 354, or 357 in rat H3receptor (Q9QYN8), similarly to mouse and human H3 receptors.

In addition, constitutively active H3 receptor mutants of the presentinvention are preferably derived from species including, but notparticularly limited to, humans, mice, rats, or guinea pigs.

Human H3 receptors in the present invention refer to H3 receptorscomprising an RDRKVAK sequence (SEQ ID NO: 11). Specific examples ofhuman H3 receptors in the present invention include, but are not limitedto, Q9Y5N1 of 445AA (SEQ ID NO: 3), BAB20090 of 453AA, and AAK50040 of365AA. In addition, mouse, rat or guinea pig H3 receptors in the presentinvention refer to H3 receptors comprising an RDKKVAK sequence (SEQ IDNO: 12). Specific examples of these include, but are not limited to,mouse H3 receptor comprising the amino acid sequence of SEQ ID NO: 1,rat H3 receptors Q9QYN8 of 445AA, BAA88765 of 449AA, BAA88767 of 413AA,and BAA88768 of 397AA, and guinea pig H3 receptor Q9JI35 of 445AA. AllH3 receptors comprising the aforementioned specific sequences arereported to comprise similar structural characteristics, activities, andactivation regulating site sequences (RDRKVAK (SEQ ID NO: 11) in humans,and RDKKVAK (SEQ ID NO: 12) in mice, rats and guinea pigs).

In addition, the sequences of activation regulating sites ofconstitutively active human H3 receptor mutants in the present inventionpreferably comprise, but are not limited to, KDHKVLK (SEQ ID NO: 4),RARKVAK (SEQ ID NO: 5), RDRKVIK (SEQ ID NO: 6) or RDRKVKK (SEQ ID NO:7). Furthermore, in the present invention, the sequences of activationregulating sites of constitutively active H3 receptor mutants in mice,rats, and guinea pigs are preferably KDHKVLK (SEQ ID NO: 4), RAKKVAK(SEQ ID NO: 8), RDKKVIK (SEQ ID NO: 9) or RDKKVKK (SEQ ID NO: 10), butare not limited to these.

Preferable examples of constitutively active mouse H3 receptor mutantsin the present invention include, but are not limited to, constitutivelyactive mutants in which the A of amino acid 357 in the amino acidsequence of SEQ ID NO: 1 is substituted with K or I; constitutivelyactive mutants in which the D of amino acid 353 is substituted with A;or constitutively active mutants in which the R of amino acid 352 issubstituted with K, the K of amino acid 354 is substituted with H, andthe A of amino acid 357 is substituted with L. For example, there arenumerous combinations of the aforementioned substitution mutations.

In addition, preferable examples of constitutively active human H3receptor mutants include, but are not limited to, constitutively activemutants in which the A of amino acid 357 in the amino acid sequence ofSEQ ID NO: 3 is substituted with K or I; constitutively active mutantsin which the D of amino acid 353 is substituted with A, or the R ofamino acid 352 is substituted with K, the K of amino acid 354 issubstituted with H, and the A of amino acid 357 is substituted with L.For example, there are numerous combinations of the aforementionedsubstitution mutations.

A constitutively active H3 receptor mutant of the present invention canbe produced by, for example, introducing a mutation into a DNA thatencodes a protein functionally equivalent to a protein that contains theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, so that theactivity of the protein is further increased.

Examples of “a DNA that encodes a protein functionally equivalent to aprotein that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 3” include DNAs that encode mutants, alleles, variants or homologuesand such of proteins that comprise the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 3. Herein, “functionally equivalent” refers to a proteinof interest comprising a biological function (role) or biochemicalfunction (property) equivalent to a protein that comprises the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In the present invention,examples of biological functions (roles) of a protein that comprises theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 includeintracellular signal transduction functions (e.g., changes in cAMPconcentration, calcium concentration, G protein activity, phospholipaseC activity, or pH), or functions that control body weight, food intake,and blood insulin or blood leptin levels. In addition, examples ofbiochemical functions (properties) of a protein that comprises the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 include the property ofbinding with histamine or analogs thereof.

Known examples of DNAs that encode such proteins include DNAs derivedfrom humans (PCT/JP99/07280, Lovenberg, T. W. et al., MolecularPharmacology, 55: 1101-1107, 1999), rats (PCT/JP99/07280, Lovenberg, T.W. et al., Journal of Pharmacology and Experimental Therapeutics, 293:771-778, 2000), guinea pigs (Tardivel-Lacombe, J. et al., MolecularNeuroscience 11: 755-759, 2000) and mice (WO 2003004637). Thesesequences have already been disclosed.

In order to prepare DNAs comprising these other sequences, those ofordinary skill in the art can prepare DNAs that encode proteinsfunctionally equivalent to proteins comprising the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO: 3, by introducing suitable mutations intoDNAs that encode proteins comprising the amino acid sequence of SEQ IDNO: 1 or SEQ ID NO: 3, using site-directed mutagenesis (Gotoh, T. etal., Gene 152, 271-275, 1995; Zoller, M. J. and Smith, M., MethodsEnzymol. 100, 468-500, 1983; Kramer, W. et al., Nucleic Acids Res. 12,9441-9456, 1984; Kramer, W. and Fritz, H. J., Methods Enzymol. 154,350-367, 1987; Kunkel, T. A., Proc. Natl. Acad. Sci. USA, 82, 488-492,1985; Kunkel, Methods Enzymol. 85, 2763-2766, 1988), double primermethods (Zoller, M. J. and Smith, M., Methods Enzymol. 154, 329-350,1987), cassette mutagenesis (Wells, et al., Gene 34, 315-323, 1985),megaprimer methods (Sarkar, G. and Sommer, S. S., Biotechniques 8,404-407, 1990) and such. In addition, amino acid mutations can alsooccur naturally. The number of amino acids that are mutated is normally30 amino acids or less, preferably 15 amino acids or less, and morepreferably five amino acids or less (e.g., three amino acids or less).

Examples of other methods, known to those of ordinary skill in the art,for producing DNAs that encode proteins functionally equivalent to agiven protein are methods using hybridization techniques (Sambrook, J.et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.Press, 1989). More specifically, those of ordinary skill in the art knowtechniques for using a DNA sequence that encodes a protein comprising anamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 (e.g., the DNA ofSEQ ID NO: 2), or a portion thereof, to isolate DNAs highly homologousto that DNA, and techniques for using these DNAs to isolate proteinsfunctionally equivalent to proteins comprising the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO: 3.

The hybridization conditions for isolating DNAs that encode proteinsfunctionally equivalent to a protein comprising the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO: 3 can be suitably selected by those ofordinary skill in the art. Low stringency conditions are an example ofhybridization conditions. Low stringency conditions are, for example,42° C., 2×SSC and 0.1% SDS during post-hybridization washing, andpreferably 50° C., 2×SSC and 0.1% SDS. More preferable hybridizationconditions are, for example, high stringency conditions. High stringencyconditions are, for example, 65° C., 0.1×SSC and 0.1% SDS. Under theseconditions, as temperature increases, DNAs comprising higher homologycan be expected to be efficiently obtained. However, a number ofelements, such as temperature and salt concentration, are thought toaffect hybridization stringency, and those of ordinary skill in the artcan achieve similar stringencies by suitably selecting these elements.

In addition, DNAs that encode proteins functionally equivalent toproteins comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:3 can be isolated by a gene amplification method such as PCR, using aprimer that is synthesized based on the sequence information of a DNAthat encodes a protein comprising the amino acid sequence described inSEQ ID NO: 1 or SEQ ID NO: 3 (e.g., the DNA of SEQ ID NO: 2).

Proteins functionally equivalent to proteins comprising the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 3, encoded by the DNAs isolatedby such hybridization or gene amplification techniques, normallycomprise amino acid sequences with high homology to a protein comprisingthe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. High homologynormally refers to identity of at least 50%, preferably 75% or higher,more preferably 85% or higher, and even more preferably 95% or higher,at the amino acid level.

The degree of identity of one amino acid sequence or nucleotide sequenceto another can be determined by Karlin and Altschul's BLAST algorithm(Proc, Natl. Acad. Sci. USA, 90:5873-5877, 1993). Programs such asBLASTN and BLASTX were developed based on this algorithm (Altschul etal., J. Mol. Biol. 215:403-410, 1990). To analyze a nucleotide sequenceaccording to BLASTN, based on BLAST, parameters are set, for example, atscore =100 and work length =12. On the other hand, parameters used forthe analysis of amino acid sequences by BLASTX, based on BLAST, include,for example, score =50 and word length =3. When using the BLAST andGapped BLAST programs, each program's default parameters are used.Specific techniques for each analysis are known in the art(ncbi.nlm.nih.gov).

In addition, DNAs that encode proteins functionally equivalent toproteins comprising the amino acid sequence described in SEQ ID NO: 1 orSEQ ID NO: 3 include cDNAs, genomic DNAs and synthetic DNAs. cDNAs canbe screened by, for example, using ³²P or such to label the cDNAdescribed by SEQ ID NO: 2, fragments thereof, their complementary DNAsor RNAs, or synthetic oligonucleotides comprising a portion of the cDNAsequence, and hybridizing these to a tissue-derived cDNA library (e.g.,brain, thalamus or hypothalamus) expressing a DNA that encodes a proteinfunctionally equivalent to a protein comprising the amino acid sequencedescribed in SEQ ID NO: 1 or SEQ ID NO: 3. Alternatively, cDNAs can alsobe cloned by synthesizing oligonucleotides that correspond to the cDNAnucleotide sequence, and amplifying by PCR using a cDNA derived from asuitable tissue (e.g., brain, thalamus or hypothalamus) as a template.Genomic DNAs can be screened by, for example, using ³²P or the like tolabel the cDNA described by SEQ ID NO: 2, fragments thereof, theircomplementary DNAs or RNAs, or synthetic oligonucleotides comprising aportion of the cDNA sequence, and hybridizing these to a genomic DNAlibrary. Alternatively, genomic DNAs can also be cloned by synthesizingoligonucleotides that correspond to the cDNA nucleotide sequence, andamplifying by PCR using genomic DNA as a template. Synthetic DNAs can beprepared by, for example, chemically synthesizing oligonucleotides thatcomprise a partial sequence of the cDNA of SEQ ID NO: 2, annealing theseto form double strands, and then ligating them with DNA ligase (Khorana,H. G. et al., J. Biol. Chem. 251, 565-570, 1976; Goeddel, D. V. et al.,Proc. Natl. Acad. Sci. USA 76, 106-110, 1979).

In the present invention, mutations that further increase proteinactivity are introduced into the DNAs encoding proteins functionallyequivalent to proteins comprising an amino acid sequence of SEQ ID NO: 1or SEQ ID NO: 3 obtained in this manner. The sites at which amino acidsare mutated due to the introduction of DNA mutations are preferablysites equivalent to at least one of positions 352, 353, 354, or 357 inthe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, however thesites are not limited to these. In addition, the types of mutationspreferably include, but are not limited to, mutations involving aminoacid substitution. More specifically, for example, types of mutationsinclude mutations involving amino acid deletion or insertion.

Those of ordinary skill in the art can use known methods to prepareconstitutively active H3 receptor mutants from the DNAs that encode theconstitutively active H3 receptor mutants obtained in this manner.

In addition, the present invention provides DNAs that encode theaforementioned constitutively active H3 receptor mutants. The DNAs ofthe present invention have preferably been isolated. Herein, “isolated”refers to the state of having been taken out of an inherent environmentand substantially purified.

Such DNAs are useful for producing recombinant proteins. Morespecifically, constitutively active mutants of the present invention canbe prepared by inserting the aforementioned DNAs that encodeconstitutively active mutants into suitable expression vectors,introducing the vectors into suitable cells, culturing the resultingtransformants, and purifying the expressed proteins. In addition, sincethe constitutively active mutants of the present invention arereceptors, they can also be prepared by expression on a cell membrane.

Specifically, if the host is Escherichia coli, plasmid vectors such aspET-3 (Rosenburg A. H. et al., Gene 56, 125-135, 1987) and pGEX-1 (SmithD. B. and Johnson K. S., Gene 67, 31-40, 1988) may be used. E. coli canbe transformed by the Hanahan method (Hanahan D., J. Mol. Biol. 166,557-580, 1983), electroporation (Dower W. J. et al., Nucleic Acids Res.16, 6127-6145, 1988), and such. If the host is fission yeast(Schizosaccharomyces pombe), a plasmid vector such as pESP-1 (Lu Q. etal., Gene 200, 135-144, 1997) can be used. Yeast can be transformed byspheroplast fusion (Beach D. and Nurse P., Nature 290, 140, 1981), andlithium acetate methods (Okazaki K. et al. Nucleic Acids Res. 18,6485-6489, 1990), etc.

If the host is a mammalian cell, such as Chinese Hamster ovary-derivedCHO cells and human HeLa cells, vectors such as pMSG (Clontech) can beused. Alternatively, in case of HEK293 cells, pcDNA3.1(+) can be used.Recombinant DNAs can be introduced into mammalian cells by the calciumphosphate method (Graham F. L. and van derEb A. J., J. Virology 52,456-467, 1973), DEAE-dextran methods (Sussman D. J. and Milman G., Mol.Cell. Biol. 4, 1641-1643, 1984), lipofection (Felgner P. L. et al.,Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987), and electroporation(Neumann E. et al., EMBO J. 1, 841-845, 1982), etc. If the host is aninsect cell, a baculovirus vector such as pBacPAK8/9 (Clontech) can beused. Insect cells can be transcribed by methods described in literature(BioTechnology 6, 47-55, 1980).

Recombinant proteins expressed in host cells can be purified by knownmethods. The proteins can also be synthesized as fusion proteins taggedwith histidine residues at the N-terminus, or fused toglutathione-S-transferase (GST), and purified using their bindingability toward a metal-chelating or GST-affinity resin (Smith M. C. etal., J. Biol. Chem. 263, 7211-7215, 1988), respectively. For instance,when the vector pESP-1 is used, the protein of interest can besynthesized as a GST fusion protein, which can then be purified usingGST affinity resin. To separate the protein of interest, fusion proteinsmay be digested with thrombin, or blood coagulating factor Xa.

In addition, the present invention also provides methods for evaluatingwhether or not a test compound changes the activity of a constitutivelyactive mutant of the present invention. In these methods, a testcompound is first contacted with cells expressing a constitutivelyactive mutant of the present invention. There are no particularrestrictions as to the test compound used in the present methods, andexamples include, but are not limited to, single compounds such asnaturally-occurring compounds, organic compounds, inorganic compounds,proteins, peptides or nucleotides, as well as compound libraries,expression products of gene libraries, cell extracts, cell culturesupernatants, microbial fermentation products, marine organism extracts,plant extracts, and extracts of tissues or cells for which ligands arepredicted to be present (such as the brain, thalamus and hypothalamus).

In addition, cells expressing a constitutively active mutant of thepresent invention can be produced by, for example, introducing cells(such as HEK293 cells) with a vector containing a DNA encoding aconstitutively active mutant of the present invention. The vector can beintroduced into the cells by ordinary methods, using calcium phosphateprecipitation, electroporation, lipofectamine, microinjection, or such.

In the present invention, “contact” can be carried out by, for example,adding a test compound to a cell culture. When the test compound is aprotein, for example, a vector containing a DNA encoding the protein canbe introduced into cells expressing a constitutively active mutant.

In the present methods, the activities of the constitutively activemutants in the cells are then detected. The activities of theconstitutively active mutants can be detected using intracellular signaltransduction (such as changes in cAMP concentration, calciumconcentration, G protein activity, phospholipase C activity, or pH) asan indicator. Those of ordinary skill in the art can use known methodsto detect the activity of a constitutively active mutant usingintracellular signal transduction as an indicator. In the presentmethods, a test compound is judged to have changed the activity of anaforementioned constitutively active mutant when the activity isincreased or decreased compared to that in the absence of test compoundcontact.

H3 receptor gene knockout mice have been found to demonstrate increasedbody weight, food intake, and blood insulin or blood leptin levels.Thus, the aforementioned compounds can be drugs for the treatment orprevention of diseases characterized by changes (increases or decreases)in body weight, food intake, and blood insulin or blood leptin levels.

In addition, using the aforementioned evaluation methods, a number oftest compounds can be screened for drug candidate compounds that changethe activity of a constitutively active mutant. Examples of such drugcandidate compounds include, but are not limited to, H3 receptoragonists, antagonists and inverse agonists (inverse agonist drugs thatbind to receptors to express an action opposite to agonistpharmacological actions). The agonists and inverse agonists in thepresent invention include not only those with complete activity, butalso those with partial activity. The screening methods of the presentinvention are more effective methods, especially for screening forvarious drug candidate compounds that are inverse agonists of H3receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequences of H3 receptors introduced withmutations. m-H3 refers to a wild type mouse H3 receptor.

FIG. 2 shows the amino acid sequences of mouse H3 receptors introducedwith mutations.

FIG. 3 shows amino acid sequences of human H3 receptors introduced withmutations. h-H3 refers to a wild type human H3 receptor.

FIG. 4 is shows the histamine-responsiveness of H3 constitutively activemutants.

FIG. 5 shows the thioperamide-responsiveness of H3 constitutively activemutants.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more specifically through thefollowing Examples, but is not limited to these Examples.

EXAMPLE 1

The internal domain 3 of seven transmembrane type G protein-coupledreceptors is important for G protein binding or receptor activity, andis well conserved. In H3 receptors, a type of G protein-coupledreceptor, this region is also conserved in the same way. An attempt wastherefore made to produce constitutively active mouse H3 receptormutants, and constitutively active human H3 receptor mutants by usingPCR to introduce point mutations into a sequence that encodes thisregion in mouse H3 receptor cDNA and human H3 receptor cDNA.

More specifically, the amino acid mutants were first designed (FIG. 1).Next, PCR was carried out using mouse H3 receptor cDNA (expressionvector: pcDNA3.1(+)) as a template, and using primers 722F (5′-AGA ACCCCC ACC TGA TGC-3′ (SEQ ID NO: 19)) and 1338R (5′-TCA CTT CCA GCA CTGCTC CAG G-3′ (SEQ ID NO: 20)), along with 683F (5′-GCA CTC GTC TTC GGCTGG ATG-3′ (SEQ ID NO: 21)) and MT1 (5′-CGA CTT GAG TAC CTT CTT GTC-3′(SEQ ID NO: 22)), MT2 (5′-CGA CTT GAG TAC CTT GTG GTC CTT CGA CAG CCG-3′(SEQ ID NO: 23)), MT3 (5′-CTT CTT GGC CCG CGA CAG CCG-3′ (SEQ ID NO:24)), MT5 (5′-CGA CTT GAT TAC CTT CTT GTC-3′ (SEQ ID NO: 25) or MT6(5′-CGA CTT CTT TAC CTT CTT GTC CCG-3′ (SEQ ID NO: 26)). 25 cycles of“94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 30seconds” were carried out. PCR was conducted again a second time, usingthe fragments obtained from each PCR reaction as templates, and usingprimers 683F and 1338R (25 cycles of “94° C. for 15 seconds, 55° C. for30 seconds, and 72° C. for 30 seconds”). The fragment produced by thesecond round of PCR (656 bp) was cloned to pCR2.1-TOPO. As a result ofinserting point mutations, the BstXI sites of MT1, MT5, and MT6, and theBsmFI sites of MT2 and MT3, were respectively deleted. The introductionof point mutations was then confirmed by sequencing. Next, anAor51HI-SfiI fragment (174 bp) comprising a point mutation was cloned tomouse H3 receptor cDNA. The Expand High-Fidelity PCR System(Boehringer-Mannheim) was used for all PCR reactions.

Insertion of the point mutation was confirmed by sequencing the mutatedDNA fragment. An Aor51HI-SfiI fragment was then cloned to wild typemouse H3 receptor cDNA (expression vector: pcDNA3.1(+)). Theaforementioned method was used to produce the MT1, MT2, MT3, MT5, andMT6 clones (FIG. 2).

Wild type mouse H3 receptor cDNA and the five mouse H3 receptor mutantcDNAs were each transfected into cell line HEK293, and screened withG418 to obtain their respective stable clones. Northern analysis wasused to check expression levels, and stable clones with roughly equalexpression levels were used in the experiment.

Constitutively active human H3 receptor mutants were produced using thesame methods (FIG. 3), and mutant-expressing clones were obtained.

EXAMPLE 2

Since H3 receptors are Gi-binding-type G protein-coupled receptors, cAMPlevels were measured using ELISA. More specifically, 10⁵ cells per wellwere cultured in a 24-well plate the day before testing. On the day ofthe test, cells were cultured for 15 minutes in the absence of serum,and then treated for 15 minutes with 0.5 mM IBMX. Forskolin (10 μM),histamine (10⁻¹¹ M to 10⁻⁶ M) and thioperamide (10⁻¹⁰ M to 10⁻⁵ M) wererespectively added, and the cells were treated for 15 minutes. The cAMPEnzyme Immunoassay (EIA) System (Amersham) was used to measure cAMP. Thecells were lysed with 150 μl of lysing reagent 1B, provided with thekit. 5 μl of the cell lysate and rabbit anti-cAMP antibody were reactedby being allowed to stand undisturbed at 4° C. for two hours on anantibody-immobilized plate. Moreover, enzyme-labeled antibody was addedand reacted by being allowed to stand undisturbed at 4° C. for one hour.The plate was washed with buffer, and enzyme substrate solution was thenadded and reacted by being allowed to stand undisturbed at roomtemperature for about 30 minutes. The reaction was stopped with 1 Nsulfuric acid, and optical absorbance was then measured. A standardcurve was produced from the optical absorbances of standard cAMPsolutions, and cAMP levels were determined. A similar test was conductedby treating the cells for 18 hours with pertussis toxin (PTX) at a finalconcentration of 100 ng/ml. As a result, in the presence of 10 μMforskolin, the cAMP levels in all clones decreased histaminedose-dependently (FIG. 4).

EXAMPLE 3

In the presence of 10 μM forskolin, the H3 inverse agonist, thioperamidewas found to increase cAMP levels dose-dependently, particularly for theMT6 clone (FIG. 5). Although H3 receptors are constitutively active evenin their natural states, and are reported to easily adopt constitutivelyactive conformations, the M6 clone was observed to show a cAMP increaseof about five-fold compared to the wild type, thus suggesting anextremely strong constitutively active state. In addition, to determinewhether the increase in cAMP level caused by thioperamide was mediatedby the Gi protein pathway, a similar experiment was conducted bytreating the cells with pertussis toxin (PTX) at a final concentrationof 100 ng/ml for 18 hours. The results showed that PTX inhibited theincreases in cAMP caused by thioperamide were in both the wild type H3and MT6 clones.

H3 receptors are present in the anterior portion of synapses, andregulate histamine release by functioning as autoreceptors. H3 receptorsare constitutively active forms, and act to reduce histamine release,even in the absence of histamine. In addition, histamine acts to furtherreduce histamine release by binding to H3 receptors.

Histamine acts to reduce appetite when it binds to H1 receptors, presentin the posterior portion of synapses. H1 agonists can serve asantiobesity drugs. However, since H1 distribution is ubiquitous, theyalso comprise actions other than the target action. On the other hand,H3 antagonists and inverse agonists can serve as antiobesity drugs sincethey increase histamine release by acting only in the central nervoussystem.

Even constitutively active forms of inverse agonists demonstrateantagonistic effects. In fact, inverse agonists have already beenindicated to be more effective than antagonists (Milligan, G. et al.,TiPS, 16, 10-13, 1995).

In the present Examples, H3 clones comprising extremely strongconstitutive activity were successfully produced by using PCR tointroduce point mutations into sequences that encode the internal domain3 of H3 receptors. The use of these clones is considered to makescreening for H3 receptor inverse agonists and such both easier and moreefficient.

INDUSTRIAL APPLICABILITY

The present inventors produced constitutively active H3 receptormutants. By using constitutively active H3 receptor mutants, drugcandidate compounds such as H3 receptor inverse agonists can be screenedmore easily and efficiently.

1. A constitutively active mammalian H3 receptor mutant, the mutantcomprising at least one mutation in internal domain 3 of the H3receptor.
 2. The constitutively active mutant of claim 1, wherein theinternal domain 3 includes an activation-regulating site on itsC-terminal side, and the at least one mutation comprises one or moreamino acid substitutions within the activation-regulating site.
 3. Theconstitutively active mutant of claim 1, wherein the at least onemutation comprises an amino acid substitution at one or more ofpositions 352, 353, 354, and 357 in the amino acid sequence of SEQ IDNO: 1 or SEQ ID NO:
 3. 4. The constitutively active mutant of claim 1,wherein the at least one mutation includes either (a) or (b) below: (a)in a human H3 receptor, replacing RDRKVAK (SEQ ID NO:11) with KDHKVLK(SEQ ID NO:4), RARKVAK (SEQ ID NO:5), RDRKVIK (SEQ ID NO:6) or RDRKVKK(SEQ ID NO:7); or (b) in a mouse, rat, or guinea pig H3 receptor,replacing RDKKVAK (SEQ ID NO:12) with KDHKVLK (SEQ ID NO:4), RAKKVAK(SEQ ID NO:8), RDKKVIK (SEQ ID NO:9) or RDKKVKK (SEQ ID NO:10).
 5. Theconstitutively active mutant of claim 1, wherein the at least onemutation comprises at least one of (a) to (c) below: (a) a substitutionfrom A to K or I at position 357 in the amino acid sequence of SEQ IDNO: 1; (b) a substitution from D to A at position 353 in the amino acidsequence of SEQ ID NO: 1; and (c) a substitution from R to K at position352, K to H at position 354, and A to L at position 357 in the aminoacid sequence of SEQ ID NO:
 1. 6. The constitutively active mutant ofclaim 1, wherein the at least one mutation comprises at least one of (a)to (c) below: (a) a substitution from A to K or I at position 357 in theamino acid sequence of SEQ ID NO: 3; (b) a substitution from D to A atposition 353 in the amino acid sequence of SEQ ID NO: 3; and (c) asubstitution from R to K at position 352, R to H at position 354, and Ato L at position 357 in the amino acid sequence of SEQ ID NO:
 3. 7. Amethod for evaluating whether or not a test compound changes theactivity of a constitutively active mammalian H3 receptor mutant,wherein the method comprises: (a) contacting the test compound withcells expressing the constitutively active mammalian H3 receptor mutantof claim 1; and (b) detecting the activity of the constitutively activemutant in the cells, wherein the test compound is judged to change theactivity of the constitutively active mutant when the activity increasesor decreases in the presence of the test compound compared with activityin the absence of the test compound.
 8. The method of claim 7, whereinthe activity of the constitutively active mutant is detected by using achange in cAMP concentration, a change in calcium concentration, achange in G protein activity, a change in phospholipase C activity, or achange in pH, as an indicator.
 9. A method of screening for a drugcandidate that changes the activity of a constitutively active mammalianH3 receptor mutant, wherein the method comprises steps (a) and (b)below: (a) using the method of claim 8 to evaluate a plurality of testcompounds to determine whether or not they change the activity of theconstitutively active H3 receptor mutant; and (b) selecting from theplurality of test compounds one or more compounds judged to change theactivity of the constitutively active mutant.
 10. A method of screeningfor a drug candidate that changes the activity of a constitutivelyactive H3 receptor mutant, wherein the method comprises steps (a) and(b) below: (a) using the method of claim 7 to evaluate a plurality oftest compounds to determine whether or not they change the activity ofthe constitutively active H3 receptor mutant; and (b) selecting from theplurality of test compounds one or more compounds judged to change theactivity of the constitutively active mutant.
 11. The method of claim10, wherein the drug candidate is an H3 receptor inverse agonist. 12.The method of claim 10, wherein the internal domain 3 includes anactivation-regulating site on its C-terminal side, and the at least onemutation comprises one or more amino acid substitutions within theactivation-regulating site.
 13. The method of claim 12, wherein the atleast one mutation comprises an amino acid substitution at one or moreof positions 352, 353, 354, and 357 in the amino acid sequence of SEQ IDNO: 1 or SEQ ID NO:
 3. 14. The method of claim 12, wherein the at leastone mutation comprises either (a) or (b) below: (a) in a human H3receptor, replacing RDRKVAK (SEQ ID NO:11) with KDHKVLK (SEQ ID NO:4),RARKVAK (SEQ ID NO:5), RDRKVIK (SEQ ID NO:6), or RDRKVKK (SEQ ID NO:7);or (b) in a mouse, rat, or guinea pig H3 receptor, replacing RDKKVAK(SEQ ID NO:12) with KDHKVLK (SEQ ID NO:4), RAKKVAK (SEQ ID NO:8),RDKKVIK (SEQ ID NO:9) or RDKKVKK (SEQ ID NO:10).
 15. The method of claim12, wherein the at least one mutation comprises at least one of(a), (b)and (c) below: (a) a substitution from A to K or I at position 357 inthe amino acid sequence of SEQ ID NO: 3; (b) a substitution from D to Aat position 353 in the amino acid sequence of SEQ ID NO: 3; and (c) asubstitution from R to K at position 352, R to H at position 354, and Ato L at position 357 in the amino acid sequence of SEQ ID NO:
 3. 16. Anisolated DNA encoding a constitutively active mutant of a mammalian H3receptor, the mutant comprising at least one mutation in internal domain3 of the H3 receptor.
 17. The DNA of claim 16, wherein theconstitutively active mutant comprises an amino acid substitution at oneor more of positions 352, 353, 354, and 357 in the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO:
 3. 18. The DNA of claim 16, wherein theconstitutively active mutant has at least one mutation selected fromeither (a) or (b) below: (a) in a human H3 receptor, replacing RDRKVAK(SEQ ID NO: 11) with KDHKVLK (SEQ ID NO: 4), RARKVAK (SEQ ID NO: 5),RDRKVIK (SEQ ID NO: 6) or RDRKVKK (SEQ ID NO: 7); or (b) in a mouse,rat, or guinea pig H3 receptor, replacing RDKKVAK (SEQ ID NO: 12) withKDHKVLK (SEQ ID NO: 4), RAKKVAK (SEQ ID NO: 8), RDKKVIK (SEQ ID NO: 9)or RDKKVKK (SEQ ID NO: 10).
 19. The DNA of claim 16, wherein theconstitutively active mutant has at least one mutation selected from thegroup consisting of (a) to (c) below: (a) a substitution from A to K orI at position 357 in the amino acid sequence of SEQ ID NO: 3; (b) asubstitution from D to A at position 353 in the amino acid sequence ofSEQ ID NO: 3; and (c) a substitution from R to K at amino acid 352, R toH at position 354, and A to L at position 357 in the amino acid sequenceof SEQ ID NO:
 3. 20. A vector containing a DNA encoding a constitutivelyactive mutant of a mammalian H3 receptor, the mutant comprising at leastone mutation in internal domain 3 of the H3 receptor.
 21. A transformedcell comprising (a) a DNA encoding a constitutively active mutant of amammalian H3 receptor, the mutant comprising at least one mutation ininternal domain 3 of the H3 receptor, or (b) a vector containing saidDNA.